ABSTRACT Influenza virus is a serious public health threat. Seasonal outbreaks cause significant morbidity and mortality, and pandemic outbreaks have the potential for widespread infection and disease. Novel pandemic viruses emerge from animal reservoirs to rapidly become the dominant circulating strains, as was the case during the emergence of the 2009 H1N1 pandemic virus. Like all viruses, influenza virus is completely dependent upon the host cell for replication. Influenza virus exploits and subverts host processes while at the same time evading cellular antiviral responses. The balance between these pro- and antiviral forces influences the outcome of a viral infection, yet we have limited knowledge on the host factors engaged during replication and how they impact disease severity and the emergence of new viruses. This information is required to define the molecular mechanisms underpinning a productive infection. The overall goal of this proposal is to identify host factors and their mechanisms that regulate influenza virus replication. We have generated data on a genome-wide scale identifying new host factors that impact infection. We specifically focused on events that occur after influenza virus enters the host cell. Results from our genome-wide knockout screen reveal that a large class of pro-apoptotic and innate antiviral factors, including IFIT2 and IFIT3, are actually important enhancers of influenza virus replication. We hypothesize that while apoptosis is generally seen as a last-resort antiviral defense, influenza virus has evolved to exploit the apoptotic cellular environment for maximal viral replication. In Aim 1, we will determine the impact of IFIT2 and IFIT3 on infection and define the functions of IFIT2 and apoptosis that promote viral replication. In Aim 2, we will use a newly developed technique to perform a virally driven selection of host modulators and use sophisticated network analysis to identify key players and pathways at the virus:host interface. We will then use the experimental strategies optimized in Aim 1 to define the mechanisms of these newly identified viral regulators. Major strengths of our strategy are that it can identify both pro- and antiviral co-factors and it is not restricted to influenza virus, but can be quickly adapted to study virus:host interactions in almost any viral system. The results from this proposal will establish a mechanistic understanding of viral and host factors regulating infection. This detailed knowledge is essential for defining the molecular events that underpin a successful infection and identifying new targets that can be exploited for the rational development of antiviral therapies.